Epstein-Barr virus (EBV) is associated with B-cell as well as epithelial cell malignancies. Like all herpesviruses, EBV infects cells in either a latent or lytic form. The lytic form of infection is required for transmission of the virus from host to host, and cell to cell. Expression of either one of the two viral immediate-early (IE) proteins, BZLF1 (Z) and BRLF1(R), in latently infected cells is sufficient to induce the lytic form of infection, in which the virus replicates using its lytic origin of replication, oriLyt. In this grant, we propose to continue our long-term studies regarding the importance of the EBV IE proteins, as well as the BMRF1 early protein, for lytic EBV replication. During the previous period of funding, we showed that the BMRF1 early protein functions not only as the viral DNA polymerase accessory protein, but also as a transcription factor that activates an oriLyt promoter (BHLF1) as well as the cellular gastrin promoter. In addition, we recently discovered that the IE Z protein is uniquely capable of preferentially binding to, and activating, certain EBV target promoters when these promoters are in the methylated form. We have also shown that Z disperses nuclear PML (ND10 bodies), and perhaps as a consequence of this, reduces the level of certain DNA repair proteins in the host cell. We propose the following specific aims. In Specific aim #1, we will dissect the mechanisms by which the BMRF1 protein functions not only as the viral DNA polymerase accessory factor, but also as a transcription factor. In Specific aim #2, we will determine whether DNA methylation of oriLyt affects either its replication, or its transcription, and vice versa if the viral proteins known to regulate oriLyt transcription (Z, R, and BMRF1) affect oriLyt methylation. In Specific Aim #3, we will determine if one or more lytic viral proteins (particularly Z, R or BMRF1) inhibit the cellular DNA repair machinery, and if this ability to inhibit cellular DNA repair is important for protecting the viral ends from concatamerization during the lytic form of infection. These studies should enhance our understanding of how the Z, R and BMRF1 proteins function not only as viral transcription factors, but also as essential viral replication proteins.